Matrix Microfluidic Device for High-Throughput Single Cell Transcriptomics
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Solution Overview
Problem
Current methods for analyzing single cells using Fluidigm IFCs are limited by the inability to easily identify transcripts from combined cDNA samples, especially when dealing with more than 96 cells, as commercial kits require addressable outlets for each cell, and sequencing methods like Illumina's bridge amplification and sequencing require individual tagmentation reactions for controlled fragmentation and sample identification.
Innovation Solution
A matrix-type microfluidic device with capture sites arranged in a matrix format, allowing separate delivery of reagents to each site, enabling independent reactions and subsequent pooling of reaction products for high-throughput transcriptome amplification and identification, using barcoding strategies to de-multiplex cells and facilitate sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If commercial kits are used to prepare cDNA from single cells, then transcriptome analysis can be performed, but individual identification of transcripts from each cell becomes difficult when analyzing more than 96 cells
Solution Approach 1:
The device divides the analysis into separate capture sites arranged in a matrix, where each site independently captures and processes individual cells or small groups of cells. This segmentation allows simultaneous analysis of many cells while maintaining individual identification through unique barcodes at each capture site.
Solution Approach 2:
The invention uses barcode sequences as information copies to identify the origin of each cell's transcriptome. These barcodes are incorporated into the cDNA during reverse transcription, creating a permanent record of which capture site (and thus which cell) each transcript came from, enabling traceability even after pooling all samples.
2Reliability
If individual tagmentation reactions are performed for each cell's cDNA, then sequencing can be controlled and samples identified, but the process becomes complex and requires custom transposons
Solution Approach 1:
The barcode incorporation is performed preliminarily during the reverse transcription step, before tagmentation and sequencing. This preliminary tagging with capture site-specific barcodes simplifies subsequent steps, as the identification information is already embedded in the cDNA, eliminating the need for complex post-tagmentation identification procedures.
Solution Approach 2:
The invention uses universal transposons that can process all cDNA samples simultaneously, rather than requiring custom transposons for each cell. The universality of the transposase enzyme and transposon design allows pooled samples to be processed together while the embedded barcodes maintain individual sample identification.
3Productivity
If cells are distributed to capture sites for independent reactions, then high-throughput analysis is enabled, but fluidic isolation of capture sites after cell distribution becomes challenging
Solution Approach 1:
The device uses a matrix arrangement of capture sites with orthogonal row and column addressing systems. This two-dimensional organization allows fluidic isolation and reagent delivery through separate row and column channels, simplifying the control of independent reactions at each capture site while maintaining high throughput.
Data Source
Figure 1A~1B
Figure 1C~1D
Figure 2
AI summary
Described herein are cell-based analytic methods, including a method of incorporating nucleic acid sequences into reaction products from a cell population, wherein the nucleic acid sequences are incorporated into the reaction products of each cell individually or in small groups of cells individually. Also described herein is a matrix-type microfluidic device that permits at least two reagents to be delivered separately to each cell or group of cells, as well as primer combinations useful in the method and device.